The invention relates to a recombinant RNA virus vector, to a process for preparing the recombinant RNA virus vector and to its use for preparing a medicament.
The state of the art discloses DNA-dependent systems for expressing proteins in higher eukaryotic cells. For example, the baculovirus system (Bishop DHL, 1990, p. 62-67, Current Opinion in Biotechnology) has been disclosed, which system can, however, only be used in insect cells.
Other eukaryotic systems, such as the Semliki Forest virus system (Liljestrom P., Garoff H., 1991, Bio/Technology 9, 1356-1361) are based on an RNA-dependent principle; they cannot be used in vivo on account of their pathogenic potential.
Other systems are based on RNA-encoded viruses, the so-called retroviruses. Expression of foreign genes in these vector systems is not simply translation-dependent; it only takes place following integration into the genomic DNA of the target cell.
The systems which have been mentioned suffer, inter alia, from the disadvantage of integrating into the genome of the respective target cell, something which can have fatal consequences. Retroviral vector systems interact as cDNA in the genome of the target cell. All the constructs which are suitable for effecting transcription have to be provided with regulatory sequences and sequences which are required for processing.
The object of the present invention is to eliminate the disadvantages of the state of the art by means of a novel vector system and to indicate a process for preparing the vector system, and also combinations and possible applications.
This object and expedient further developments are achieved by the features of the claims.
According to the invention, a recombinant RNA virus vector for exerting an effect on cells, in particular for gene therapy, is envisaged whose sequence is altered by removing at least one sequence segment which is necessary for replication competence, and by inserting at least one foreign sequence segment, such that expression of a sequence which is different from the wild type virus can be achieved, can be mediated or can be impeded.
The advantage of this system is, in particular, that no transcription-regulating sequences are required. The novel vector system does not inevitably result in lethal damage to the infected cells and is, on account of its property of primarily transient expression, suitable for in-vivo or ex-vivo administration to a patient.
Suitably, at least one helper cell, helper cell line or helper virus is envisaged. This makes it possible to compensate for the loss in the function of the recombinant RNA virus vector which is caused by removing of at least one sequence segment which is necessary for replication competence. The sequence which is different from the wild type virus can be an antisense sequence or ribozyme sequence for blocking or destroying a viral RNA or DNA molecule.
One embodiment envisages that the sequence is derived from a positive-strand RNA virus, preferably a picornavirus, and that a poliovirus, preferably an attenuated poliovirus, is used as the picornavirus.
This ensures, with high transfection efficiency, that specific RNA sequences are expressed in the cytoplasmic region.
A part of the coding vector sequence, preferably the structural protein-encoding sequence, can be removed and the foreign sequence can be a protein-encoding sequence, in particular a cDNA sequence. In this context, it is advantageous if the recombinant RNA virus vector possesses several internal ribosomal binding sites, preferably those of encephalomyocarditis virus, of poliovirus, of foot and mouth disease virus or of hepatitis A virus.
The helper virus can be capable of being expressed transiently or stably in eukaryotic cells. It is expedient to derive the helper virus from poliovirus.
In one embodiment of the recombinant RNA virus vector, a "suicide gene" can be prepared contrary to the reading direction of the RNA virus vector, and a combination with such sequence elements is envisaged which makes it possible to treat retroviral cells, such as HIV provirus-harboring cells, when a reverse transcriptase and, in particular, a "primer binding site" are present.
According to another solution according to the invention, a process for preparing a novel recombinant RNA virus vector is envisaged in which at least one sequence segment which is necessary for replication competence is removed from the sequence of a preferably double-stranded copy of a wild type virus, at least one foreign sequence segment is inserted and the recombinant RNA virus vector is introduced, for supplementation, into a production cell.
Advantageously, the production cell is a helper cell or a helper cell line. In this case, a helper virus can be introduced into the production cell. It is advantageous if the recombinant RNA virus vector is cloned downstream of a transcription site of a eukaryotic expression vector or downstream of a prokaryotic promoter.
In another embodiment used in the process, the recombinant RNA virus is provided by the helper cell line with an infectious coat, or the helper virus in the production cell supplies an infectious coat.
According to another solution of the object, a kit or compilation of means for implementing the novel process is supplied.
The novel recombinant RNA virus vector can be used to prepare a medicament for transient gene therapy and for treating tumor or autoimmune diseases and for treating wounds. It is also possible to treat retrovirally infected cells, in particular HIV provirus-harboring cells. The use of the novel recombinant RNA virus vector for preparing a medicament for the oral administration of proteins and/or nucleic acids is also of importance.
Since it is not possible to administer the abovementioned medicaments straightforwardly, the invention envisages a compilation of means for administering the medicaments.
A preferred use of the invention is the preparation of clones which encode an immune system-activating gene and which are suitable for being employed as medicaments for the gene therapy of tumor patients. In this context, immunomodulatory substances are to be understood as being interleukins, such as IL-2, IL-10, GMCSF and GCSF, and also immunologically relevant adhesion molecules, such as CD2, CD4, CD8 LFA-1, 4F2, CD40 and CD28, and also their ligands LFA-3, ICAM-1, B7 or CD45. These genes are preferably expressed in recombinant polio viruses in place of the structural proteins which are specific to the virus. The virus proteins which are required in cis are still expressed by using a signal, i.e. an internal ribosomal entry site (IRES), which permits polycistronic consecutive ordering of genes and their expression.
A process for transient gene therapy uses a novel recombinant RNA virus vector for exerting an effect on a host cell, with the expression of a sequence which is different from the wild type virus being achieved, mediated or impeded. Another solution to the above object is achieved by the provision of a kit or a compilation of means for implementing the process.
Expression of immunostimulatory substances in accordance with the invention makes it possible to implement cancer therapy ex vivo and in vivo; the cloning of suicide genes makes it possible to ablate particular cell types, e.g. virus-infected cells in the case of infection with HIV.
One application of the invention for treating autoimmune diseases is the preparation of clones which encode an apoptosis-inducing gene, e.g. the receptor fas, the tumor-suppressing gene p53 or the early adenovirus gene E1A, or encode a so-called suicide gene, and whose surface properties are preferably altered such that they bind especially to cells of the immune system which recognize a particular autoantigen or its epitope and infect these cells.
The use of TNF.alpha., which also elicits apoptotic and necrotic cell death, and TK and CDD, which are known as suicide genes, also aims in this direction. Particular immunologically relevant cells can also be eliminated by inducing anergy by means of depleting primary receptor molecules (e.g. using antisense constructs) or the receptor-associated motifs (Reth motifs). Specificity for target cells of this nature can be achieved by replacing the main antigen of VP1 with the reaction-triggering antigen or by means of specific receptor recognition. In the case of CD4-carrying target cells, the appropriate HIV virus epitope (gp120-V3) is an example of a possible option. Clones of this nature can be administered, preferably systemically, to autoimmune-reactive patients and can invade the autoimmune-reactive cells.
After the cells have been infected, a suicide gene or an apoptosis-inducing gene is expressed in them. This results in higher specificity for the target cells.
Another preferred application of the invention uses genes which lead to the death of the target cells. These genes additionally contain a given specificity for a target cell group, e.g. CD4 or CD26-carrying cells, as a therapeutic agent against an already existing HIV infection.
The destruction of potential HIV virus target cells and of cells into which the HIV virus has integrated leads, particularly in the phase where the virus burden is low, to remission of the infection.
Another preferred application is the expression of anti-inflammatory molecules in the therapy of autoimmunopathies which are in part tissue-destructive.
The process for preparing the novel recombinant RNA virus vector can, in particular, have the following steps:
In a first step, a double-stranded cDNA copy of an RNA virus, preferably an attenuated poliovirus, is provided, prepared, isolated or otherwise obtained (vector DNA). This cDNA copy is preferably present in a prokaryotic replication system. PA1 In a second step, regions of the cDNA construct are excised in accordance with the size of the gene to be cloned. These regions are first and foremost sequences whose function can be replaced in trans, preferably encoded coat proteins, in the case of the poliovirus the genes VP4, VP3, VP2 and/or VP1. The sequences which are required for expression in cis, the protease recognition sites which are required for processing, the internal ribosomal binding sites and the polymerases are retained. PA1 Trans-supplementation with the removed genes is ensured by means of a helper virus or a helper cell line. PA1 The gene which is to be newly expressed in the viral genome is preferably cloned downstream of a suitable internal ribosomal binding site (internal ribosomal entry site, IRES) and provided with its own methionine. The gene to be cloned preferably ends with a stop codon and possesses a further IRES for reinitiating the translation of the subsequent proteins of the virus and a methionine for resuming the translation. The newly added sequence, including the structures just mentioned, usually has about the same sequence length as the fragment which was removed. PA1 The trans-supplementing fragments are made adequately available from a helper virus or, preferably, from a helper cell line, with the expression of toxic products, for example the complete P1 fragment, usually being avoided or restricted. PA1 In one embodiment, the replication-defective recombinant poliovirus genome in a eukaryotic expression vector is transferred into the production cell and expressed in the cell. The recombinant poliovirus RNA can also be present in the production cell following in-vitro transcription and transfection, e.g. electroporation. The recombinant poliovirus RNA is packed into infectious particles in the production cell. PA1 As an alternative to this, a helper virus can also be used which, due to missing genome regions, can no longer be packed into viral particles. In this context, recombination of the helper virus and the recombinant expression virus is preferably to be avoided. PA1 The infectious particles which contain the recombinant expression virus are preferably isolated from the supernatant of the cell culture or from the cytoplasm of infected cells. PA1 The infectious particles, containing the recombinant virus genome, are employed for infecting target cells. In this context, it can be a matter of cells which are maintained in cell culture, of clones, of ex-vivo explants or of systemic or tissue-specific administrations of whole organisms.